Advances in an In Vitro Tuberculosis Infection Model Using Human Lung Organoids for Host-Directed Therapies

The emergence of drug-resistant Mycobacterium tuberculosis (M.tb) has led to the development of novel anti-tuberculosis (anti-TB) drugs. Common methods for testing the efficacy of new drugs, including two-dimensional cell culture models or animal models, have several limitations. Therefore, an appropriate model representative of the human organism is required. Here, we developed an M.tb infection model using human lung organoids (hLOs) and demonstrated that M.tb H37Rv can infect lung epithelial cells and human macrophages (hMφs) in hLOs. This novel M.tb infection model can be cultured long-term and split several times while maintaining a similar number of M.tb H37Rv inside the hLOs. Anti-TB drugs reduced the intracellular survival of M.tb in hLOs. Notably, M.tb growth in hLOs was effectively suppressed at each passage by rifampicin and bedaquiline. Furthermore, a reduction in inflammatory cytokine production and intracellular survival of M.tb were observed upon knockdown of MFN2 and HERPUD1 (host-directed therapeutic targets for TB) in our M.tb H37Rv-infected hLO model. Thus, the incorporation of hMφs and M.tb into hLOs provides a powerful strategy for generating an M.tb infection model. This model can effectively reflect host-pathogen interactions and be utilized to test the efficacy of anti-TB drugs and host-directed therapies.


Introduction
Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), is one of the most prevalent bacterial infectious diseases worldwide [1].Drug resistance presents an urgent and challenging obstacle in the treatment of TB.To address these issues, it is crucial to establish an appropriate model for the development of novel anti-TB drugs.
The primary sites of M.tb infection are the lungs, with transmission commonly occurring through the alveolar sacs following inhalation.M.tb primarily targets macrophages, particularly resident alveolar macrophages (AMs), which leading to the formation of granulomas comprising various immune cells [2].During infection, the alveolar epithelium serves as a harbor for bacterial recognition and uptake, directly interacting with AMs to regulate cytokine expression in response to pathogens [3].Alveolar epithelial cells (AECs) also provide a niche for M.tb replication and dissemination during infection [4].
The treatment and diagnosis of susceptible TB have remained unchanged over the last 40 years.In 2019, a new regimen, combination therapy with bedaquiline and linezolid, was approved to treat adults with drug-resistant TB [5]; however new drugs and treatments are still warranted as the emergence of drug-resistant TB continues to increase.In vitro cell culture models using mouse macrophages and in vivo animal models are commonly used to investigate the pathology and pathogenesis of TB and screen new drugs [6][7][8].Despite the wellknown similarities between mice and humans, these systems have several limitations, such as variations in the lung lobe structure and lung cell composition [9].Furthermore, as animals are not natural reservoirs for M.tb, they only partially mimic the clinical symptoms and immunological indicators of TB, exhibiting challenges in granuloma formation and differing susceptibilities to TB compared to those observed in humans [10].Therefore, the evaluation results using animal models for the development and application of new TB treatment drugs are less reliable.To address these limitations, it is imperative to develop a novel model utilizing human-derived cells for anti-TB drug testing.
Recently, infection models have been developed to mimic the complexity of the lung microenvironment, offering an alternative to immortal cell lines of lung epithelia and animal models [10,11].Several models have been developed using advanced cell culture systems to prevent or treat infections.For viral [12] and M.tb [13] infections, a lung-on-chip model was developed that could regulate the dynamic flow and mimic breathing-like movements.Additionally, host immune responses to Mycobacteria infection were studied using human bronchiolar airway organoids [14].Furthermore, an M.tb infection model comprising primary human blood mononuclear cells was developed using a microfluidic plate [15].In another study, in vitro formation of granuloma-like cell aggregates was achieved by utilizing peripheral blood

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Tuberculosis infection model using human lung organoids for host-directed therapies mononuclear cells isolated from patients with latent TB infection and beads coated with purified protein derivatives to mimic in vivo behavior of granuloma [16].
Although advanced cell culture systems offer advantages in modeling infectious diseases, they fail to accurately replicate the three-dimensional (3D) structure of the lungs.Human lung organoids (hLOs), which replicate the cellular composition and function of the lungs, have emerged as outstanding models for investigating pathophysiology and drug screening.For establishing severe acute respiratory syndrome coronavirus 2 infection models, human distal lung organoids consisting of AEC2 and basal cells were generated [17].However, lung models employing human-derived cells for anti-TB drug testing have not been fully developed.
In this study, we generated 3D hLOs derived from human pluripotent stem cells (hPSCs) that exhibited morphological similarities to the alveolar sacs in lungs.Subsequently, we established an in vitro M.tb infection model using hLOs (lung alveolar epithelium) and human macrophages (hMφs; immune cells) to more closely mimic the in vivo microenvironment.Additionally, we explored the potential of host-directed therapy (HDT) by regulating host genes using small interfering RNAs (siRNAs) to effectively treat intracellular M.tb in hLOs.

Generation of hPSC-derived 3D lung organoids
To assess the long-term efficacy of the anti-TB drugs, we generated 3D lung organoids derived from hPSCs following a previously established protocol (Fig 1A) [18][19][20].Briefly, hPSCs were differentiated into definitive endodermal (DE) cells, which were then re-plated onto Matrigelcoated plates and cultured in an anterior foregut endoderm (AFE) differentiation medium.Following AFE differentiation, the cells were embedded in Matrigel droplets and cultured for an additional 60 days, with passages every 7-9 days in LO medium to establish hLOs.hLOs were successfully formed and exhibited a structure with a hollow lumen similar to that of the alveolar sacs (Fig 1B).Real-time PCR (qPCR) data showed that the expression of lung progenitor cell marker NKX2.1 gradually decreased, while that of mucus-secreting cell (MUC1), alveolar type 1 epithelial cell (AEC1; HOPX), and alveolar type 2 epithelial cell (AEC2; SFTPB) markers gradually increased as hPSCs differentiated into hLOs (Fig 1C).Immunofluorescence staining also showed the expression of distal lung markers in hLOs, such as MUC1, SOX9, SFTPC, and HOPX [21][22][23], confirming the successful generation of multicellular 3D hLOs containing mucus-secreting cells, lung progenitor cells, AEC1, and AEC2 from hPSCs (Fig 1D).Furthermore, we found that small airway epithelial cell markers (MUC5AC, goblet cell; SCGB1A1, club cell; p63, basal cell) were expressed in a small proportion of hLOs, indicating that they are mainly composed of alveolar type cells (S1 Fig).

Establishment of an M.tb infection model using hLOs containing hMφs
As shown in Fig 1 and 3D hLOs comprising human lung ACEs were successfully established.To replicate the M.tb-infected lung tissue environment, we aimed to create a model that enables structural interactions between macrophages and M.tb within the hLO lumen.Our previous study [19], confirmed the presence of macrophages in hLOs.However, owing to the low proportion of macrophages for verifying immune response, we micro-injected macrophages into hLOs to achieve an appropriate proportion.Initially, we differentiated green fluorescent protein (GFP)-expressing human monocytes into hMφs with phorbol myristate acetate (PMA)/Ionomycin (S2A Fig) .FACS analysis revealed robust expression of macrophage-specific markers CD14, CD209, CD206, CD11b, CD36, and CD68 (S2B Fig) [24,25].In addition, cell aggregation indicating phagocytosis was observed under a fluorescence microscope, and there was an increase in gene expression and secretion of pro-or anti-inflammatory

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Tuberculosis infection model using human lung organoids for host-directed therapies cytokines following infection of hMφs-GFP with H37Ra-red fluorescent protein (RFP) (S2C-S2E Fig) .H37Rv-RFP and hMφs-GFP were micro-injected into the established hLO lumen (Fig 2A and S1 Video), and interestingly, H37Rv-RFP was observed to be surrounded by hMφs-GFP in the hLO lumen within 6 h after micro-injection (Fig 2B).Subsequently, we examined the immune response of hMφs-GFP-containing hLOs to H37Rv-RFP infection via qPCR analysis and enzyme-linked immunosorbent assay (ELISA).The results indicated a significant increase in the mRNA and protein expression levels of the inflammatory cytokines IL-6, IL-10, and TNF-α 48 h after H37Rv-RFP infection compared with levels in the controls (hLO alone, M.tb-infected hLO, and hLO containing hMφs) (Fig 2C and 2D).These results suggest the successful generation of an M.tb infection model using hLOs containing hMφs.

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Tuberculosis infection model using human lung organoids for host-directed therapies

Long-term culture of M.tb-infected hLO model
Owing to their short survival period, current in vitro models of M.tb infection using hMφ cell lines have limitations as preclinical drug efficacy evaluation models.These results prompted us to develop a new long-lived infection model for assessing the efficacy of anti-TB drugs in the treatment of latent TB infection.To address the fate of M.tb-infected hMφs, we have measured cell cytotoxicity and viability of hMφs after H37Rv infection (MOI = 1) for 96 hours.

Assessment of anti-TB drug efficacy using the long-lived M.tb infection model
In vitro drug testing is crucial to predict drug efficacy in clinical trials.Therefore, it is necessary to determine whether our M.tb infection model is suitable for evaluating the efficacy of anti-TB drugs as a preclinical model.We treated the M.tb infection model with the anti-TB drugs rifampicin (RIF) and bedaquiline (BDQ) for 48 h (Fig 4).We observed that the mRNA expression level and protein production of cytokines such as IL-6, IL-10 and TNF-α increased upon M.tb H37Rv infection and significantly decreased following treatment with RIF and BDQ, respectively (Fig 4A and 4B).Additionally, treatment with RIF and BDQ effectively reduced intracellular M.tb survival by 98.6 and 91.3%, respectively (Fig 4C and 4D).To assess the suitability of our M.tb infection model for long-term efficacy testing, H37Rvinfected hLOs were passaged four times every 7 days and treated with RIF (20 μg/mL) and BDQ (5 μg/mL) by adding prepared dilution to the cell culture media for 48 h before CFU assay (Fig 5A

Evaluation of the efficacy of HDT using the M.tb infection model
Next, the efficacy of HDT in removing intracellular M.tb was evaluated in the established hLOs.We have previously reported that the knockdown of HERPUD1 and MFN2 suppressed intracellular survival of M.tb in macrophages.Knockdown of MFN2 inhibited M.tb growth by disrupting the mitochondrial network, leading to apoptosis [26], while depletion of HERPUD1 resulted in increased ROS levels, leading to autophagy induction and decreased intracellular  Furthermore, we evaluated the intracellular number of M.tb after combination therapy with anti-TB drugs and siRNAs.As expected, the intracellular survival of M.tb significantly decreased when combination therapy was administered compared to that of the control group (anti-TB drug treatment alone and siRNA transfection alone) (Fig 6H).Although these findings represent the initial trials of HDT in hLOs, they suggest that our infection model with PSC-derived hLOs and monocyte-derived hMφs holds promise as a method for anti-TB drug testing and HDT approaches.

Discussion
As M.tb infection progresses, continuous recruitment and aggregation of immune cells, including macrophages, leads to granuloma formation [2,28].Although M.tb can infect and replicate within alveolar epithelial type II cells [29][30][31] and alveolar macrophages are important as early predators, the recruited monocytes and monocyte-derived macrophages play a protective role during M.tb infection [32].Considering the significant role of macrophages during infection, in this study, we used PMA-differentiated THP-1 cells as hMφs to establish hLOs.After H37Ra-RFP infection, hMφs expressed inflammatory cytokine genes, and hMφs surrounding macrophages infected with M.tb H37Ra-RFP were observed (S2 Fig) .In this study, we found that M.tb-infected hLOs were maintained after several passages.Moreover, the intracellular growth of M.tb during the long-term culture of hLOs was predicted due to the invasion of M.tb into epithelial cells and its survival inside the epithelial cells over a few passages.It seems that epithelial cells in hLOs support M.tb replication all along the 4th passages, since hMφs died within 96 hours after M.tb infection based on S5 Fig. Therefore, in the early time of infection, macrophages phagocytize M.tb and die within 96 hours during intracellular M.tb replication.When macrophages die, M.tb infection spreads to epithelial cells and replicates in the cells of hLOs.
Considering the challenges in TB modeling to accurately replicate the in vivo microenvironment, it is essential to co-culture epithelial and immune cells.For modeling M.tb infection, a lung-on-chip model involving co-culture of mouse epithelial cells, endothelial cells, and Mφs has been employed [13]; however, this model lacks a 3D structure that can replicate the in vivo lung environment.Another study attempted to co-culture human monocyte-derived macrophages with human adult stem cell-derived airway organoids (hAOs) containing Mycobacterium bovis BCG [14].Nonetheless, this co-culture system also fails to mimic the in vivo environment, as macrophages cannot traverse the basal side to access the lumen of hAOs and remove BCG.Here, we demonstrated that micro-injecting H37Rv-RFP and hMφs-GFP into the lumen of hLOs may be more efficient for investigating pathogen-host interactions in a lung-like environment.

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Tuberculosis infection model using human lung organoids for host-directed therapies environment.Our system also has limitations in that hLOs need to encompass macrophages, and other types of immune cells to accurately replicate granuloma formation.
As organoids consist of complex and diverse variables, including cell state and cell type, achieving uniform organoid production is crucial for reducing variation and enhancing standardization.In this study, we employed a cell counting-based approach to seed hLOs and analyzed each well as a single reaction, thereby minimizing the variability between wells.The organoid production method, which relies on cell counting, effectively minimizes variations among wells, rendering it suitable for the comparative evaluation of the efficacy of anti-TB drug testing.
The M.tb infection hLO model we constructed offers several advantages over the infection models presented in previous studies [14,35].First, our hLO-based M.tb infection model maintained its 3D shape, even after a single infection, for at least four passages during longterm culture.Second, the number of M.tb within the hLOs remained consistent after longterm passage, indicating the suitability of this model for drug testing.Although another study demonstrated the long-term survival of Mycobacterium abscessus (M.ab) in hAOs for 21 days [14], their hAO infection model was less suitable for drug testing due to the absence of passaged cultures of hAOs infected with M.ab, unlike our infection model.Since the long-lived M.tb infection model presented in this study can be maintained for up to 31 days, it is possible to analyze the anti-TB effect of new anti-TB drugs and determine whether anti-TB drugs can solve the problem of M.tb relapse.Third, our M.tb infection model can produce a minimum of 216 daughter hLOs, each containing a similar number of M.tb, after four passages during longterm culture with a single infection.We observed that treatment with anti-TB drugs significantly reduced intracellular M.tb survival and M.tb-induced inflammatory cytokine levels in hLOs.M.tb stimulation induces the production of inflammatory cytokines, which are a marker of disease activity and inflammation in TB [36].Measurement of inflammatory cytokines is crucial as it serves as an indicator by which treatment efficiency can be assessed [37,38].
Our infection system is a promising method for evaluating the efficacy of novel drugs and HDT.Currently, numerous HDT approaches are available for TB therapy, including enhancing autophagy, promoting phagosome maturation, inhibiting mTOR, inhibiting inflammation or necrotic cell death, and cytokine therapy [39].However, most of these approaches have been investigated in two-dimensional cell models, mice, and clinical trials.In this study, we demonstrated the use of hLOs as a model for drug testing against M.tb infection using several host-specific siRNAs targeting HDT candidates, such as MFN2 and HERPUD1 [26,40].We have previously reported that the knockdown of MFN2 inhibited the growth of M.tb by disrupting the mitochondrial network, leading to apoptosis [26] and reduction of intracellular M. tb.Previously, we reported that the knockdown of HERPUD1 also contributes to in reducing the number of M.tb by increasing ROS-mediated autophagy within macrophages [27].Consistent with our previous findings, the knockdown of HERPUD1 and MFN2 inhibited intracellular M.tb growth and reduced the production of inflammatory cytokines.This reduction in cytokines production by HDT and anti-TB drug treatment in the M.tb-infected hLO model can be considered as an indicator of the reduction of intracellular M.tb survival in host cells.Our findings are consistent with previous reports showing decreased inflammatory cytokines levels during successful TB cure with anti-TB drugs [37,38,41].Additionally, HDT strategies offer dual advantages in controlling TB by enhancing the efficacy of anti-TB drugs and by reducing tissue inflammation and disease pathology [42,43].
These findings suggest that the M.tb infection model we developed using PSC-derived hLOs and monocyte-derived hMφs is useful for evaluating TB treatment using HDT.In conclusion, M.tb H37Rv can grow in hPSC-derived hLOs for at least 4 weeks, making our M.tb infection model suitable for testing the efficacy of anti-TB drugs and HDT that require a prolonged therapy period.

Ethics statement
H9 cell line was obtained from the WiCell Research Institute (Madison, WI, USA).This study was approved by the Public Institutional Review Board of Ministry of Health and Welfare (Approval number: #P01-202104-41-001).

Maintenance of human pluripotent stem cells
hPSCs were maintained as follows: H9 cells were cultured in mTeSR1 (STEMCELL Technologies, Vancouver, Canada) on hESC-qualified Matrigel (Corning Inc., Corning, NY, USA)coated tissue culture plates (Thermo Fisher Scientific, Waltham, MA, USA) in a humidified 5% CO 2 incubator at 37˚C.H9 cells were passaged every 3-4 days using 0.5 mM ethylenediaminetetraacetic acid, and the medium was replaced with fresh mTeSR1 daily.

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Tuberculosis infection model using human lung organoids for host-directed therapies S1 Data.The value for graphs.(XLSX) S2 Data.Western blot raw and uncropped images.(PDF)

Fig 3 .
Fig 3. Establishment of a long-lived M.tb infection model using hLOs.(A) Schematic of the experimental time course.hMφs and H37Rv were micro-injected after 6 days of hLO seeding.CFU assays were performed on days 3, 10, 17, and 24 post-infection, and hLOs were passaged every 7 days at a 1:6 ratio.(B) Microscopy images of hLOs micro-injected with hMφs and H37Rv.A magnified image of the red box indicates the existence of hMφs and M.tb H37Rv in hLOs.Scale bar, 200 μm.(C) Determination of M.tb H37Rv viability in hLOs via CFU assay after 7 days of each passage.The experiments were repeated at least three times.Statistically significant differences were determined using an unpaired two-tailed t-test.****p < 0.0001 and ***p < 0.001.https://doi.org/10.1371/journal.ppat.1012295.g003